The present invention relates to expression vectors that contain hybrid ubiquitin promoters. The promoters are useful, among other uses, for high and sustained transgene expression in in vivo and ex vivo gene therapy and for recombinant protein expression in vitro.
Ubiquitin is an abundant, small, 76 amino acid protein that is expressed in all eukaryotic cells (Ciechanover et al. 2000; Wilkinson et al, 2000). The protein covalently attaches to abnormal, misfolded or short-lived proteins, marking them for destruction in proteasomes (Ciechanover, supra). Ubiquitin also associates with histones and may play a role in the regulation of gene expression (Spencer and Davie, 1999). The coding sequence is remarkably conserved evolutionarily, being identical from insect to man. There are at least three known ubiquitin genes in humans, named UbA, UbB, and UbC, which appear to contain one, three or nine precise direct repeats of the 76 amino acid coding unit, respectively (Baker and Board, Nucleic Acids Research, 15:443-463 (1987); Lund et al. 1985; Nenoi, et al 1996; and Wiborg et al., EMBO J., 4:755-759 (1985). The human UbB and UbC genes have been sequenced and shown to contain no introns within their coding regions, but each contain an intron in the 5xe2x80x2 flanking region (Baker and Board, supra; Nenoi supra). The UbC promoter has been shown to provide high level, ubiquitous expression when inserted into transgenic mice and when incorporated into plasmid DNA vectors (Johansen et al., FEBS 267:289-294 (1990); Schorpp et al., Nucleic Acids Research, 24:1787-1788 (1996); Wulff et al., 1990).
The promoter from human cytomegalovirus (CMV) (see U.S. Pat. Nos. 5,849,522; 5,168,062) is known to provide strong constitutive expression of transgenes at high levels. However, in gene therapy applications, expression levels achieved using the CMV promoter have been shown to be significantly reduced over time.
Accordingly, there remains a need to provide improved regulatory elements that are able to provide high and sustained expression of transgenes in applications such as gene therapy.
The present invention provides hybrid regulatory regions using elements from one or more ubiquitin promoters together with one or more strong enhancers. The present invention further provides DNA vectors that provide for high and persistent expression of associated coding sequences.